The present invention relates to a fluorescent detection system, which detects a fluorescent signal from a specific substance included in a sample and determines the quantity of the specific substance based on a volume of the detected fluorescent signal, in particular a fluorescence detection system, which is effective to carry out real-time monitoring (tracing changes in the volume of the fluorescent signal with time) on many samples in the field of clinical diagnosis wherein incubation at a certain temperature is required for, e.g., enzymatic reaction.
For example, in the case of carrying out real-time monitoring on the progress in generation of a fluorescent reaction product caused by enzymatic reaction, it is necessary to carry out fluorescence detection while incubating a sample (reaction liquid) at a certain temperature. In addition, many samples need to be simultaneously and rapidly dealt with in some cases as in the field of clinical diagnosis.
As systems proposed to solve the problems, there are scanning type fluorescence detection systems disclosed in JP-A-2000-088752, JP-A-2000-214090 and JP-A-2001-091463.
The scanning type fluorescence detection system disclosed in JP-A-2000-088752 is configured to have sample containers provided so as to spread along an arc, have the ring portion of a ring type optical guide provided so as to confront the sample containers in the vicinity of the sample containers with a partition plate being provided between the ring portion and the sample containers, and have the partition plate fixedly provided with an optical unit for excitation light and an optical unit for fluorescent light as shown in FIGS. 5A and 5B, whereby the partition plate, the optical unit for excitation light and the optical unit for fluorescent light are integrally rotated to individually pick up fluorescent signals and transmit the fluorescent signals to an optical sensor through the ring type optical guide.
The scanning type fluorescence detection system disclosed in JP-A-2000-214090 is configured to have sample containers provided so as to spread along a plurality of arcs, have the ring portion of a ring type optical guide provided so as to confront the sample containers with a partition plate being provided between the ring portion and the sample containers, and have the partition plate fixedly provided with an optical unit for excitation light and an optical unit for fluorescent light including at least one optical guide as shown in FIG. 6, whereby the partition plate, the optical unit for excitation light and the optical unit for fluorescent light are integrally rotated to individually pick up fluorescent signals and transmit the fluorescent signals to an optical sensor through the ring type optical guide.
The scanning type fluorescence detection system disclosed in JP-A-2001-091463 is configured to have sample containers provided so as to spread along an arc as shown in FIGS. 7A and 7B, have a partition plate fixedly provided with a small-sized excitation light source, an optical unit for excitation light and an optical unit for fluorescent light in integrally rotatable fashion, and have the fluorescent light outgoing end of an optical guide of the optical unit for fluorescent light provided on the rotational center axis so as to confront an optical sensor, whereby fluorescent light from the respective sample containers is individually picked up for detection.
When the conventional fluorescence detection systems are utilized to carry out real-time monitoring on changes in fluorescent signals from a specific substance included in samples with time while incubating the samples at a certain temperature, the following problems have been raised.
When an excitation light source having a small size and a low output is utilized to make the entire system further smaller in each of the scanning type fluorescent detection systems of JP-A-2000-088752 and JP-A-2000-214090, the fluorescent signals become too feeble to provide sufficient sensitivity even in a supersensitive sensor, such as a photo-multiplier. The reason is that the entrance of the ring type optical guide for transmission of fluorescent signals is normally as narrow as hundreds micrometer, causing the efficiency of fluorescent signals to lower. In particular, the scanning type fluorescence detection system of the JP-A-2000-214090 is likely to have insufficient sensitivity to extremely feeble fluorescent light since additional optical guides, which rotate, are provided in series with the stationary ring type optical guide to mediate the transmission of signals between the entrance and the ring type optical guide. Although the use of an excitation light source having a high output, such as an argon ion laser, can solve the insufficiency in sensitivity, a combination with a control source requires a large space, which is a bar to reduction in the physical size of the system.
Although the scanning type fluorescent detection system of JP-A-2001-091463 can solve the problem of reduction in the size, another problem is created when the number of samples to be simultaneously detected is large. The problem is caused by the followings: A reagent is added to many prepared samples one after another, and the samples with the reagent added thereto, and the samples are set in a conventional fluorescent detection system. In order to prevent external light from entering, the conventional fluorescent detection systems can not be operated until a shade cover is closed after the final sample has been set.
In particular, when the progress in incubation is rapid, a serious problem is raised. Specifically speaking, when the progress in incubation of samples is rapid, and when the number of samples to be simultaneously detected is large, it is become impossible to carry out real-time monitoring on the incubation since the incubation of a sample set at an early stage has been completed when the fluorescence detection system is operated. For this reason, even when the number of samples to be set in a single fluorescent detection system is determined as, e.g., n in design, there has been created a problem in that only far less than n number of samples can be used in practice.
This problem can be solved by eliminating the shade cover, which is provided in many fluorescence detection systems to cover the entire systems or all samples to be measured at one time for preventing external light from entering the fluorescence detection systems, or by enabling fluorescent measurement in an open state. Hereinbelow, the fluorescence measurement wherein the measurement is carried out without a shade cover for covering the entire system or all samples to be measured at one time is called xe2x80x9copen fluorescence measurementxe2x80x9d for convenience of explanation. A method for open fluorescence measurement is to provide shading caps for shading respective sample containers in a number equal to the number of the sample containers. However, when shading caps are provided to shade respective sample containers in a fluorescence detection system for measuring many samples, another problem, such as an increase in size and a cost rise, is created.
As explained, the fluorescence detection system for carrying our real-time monitoring on a fluorescent signal, in particular, carrying our real-time monitoring while incubating samples at a certain temperature needs to meet the requirements of, e.g., (a) temperature control with high accuracy, (b) rapid treatment of many samples, (c) high sensitivity, (d) high reliability (a reduce in mechanical trouble typified by disconnection or malfunction in movable parts, improvement in reproducibility of fluorescence detection, and a reduction in possibility of carry-over), (e) cost reduction (simplification in the structure of the system, and disuse of expensive parts for data treatment), and (f) a reduction in the physical size of the system, and further to realize (g) the open fluorescence detection to solve the problem deriving from a decrease in the incubation time.
It is an object of the present invention to provide a fluorescence detection method and a fluorescence detection system, which meet the requirements as stated above, in particular, to provide a system and a method for detecting fluorescence, which are effective to carry out real-time monitoring on many samples in rapid incubation fashion one after another, and which are capable of using a small-sized and supersensitive optical unit for fluorescence analysis to carry out the open fluorescence measurement.
The fluorescence detection method according to a first aspect of the present invention, which is provided to attain the object, is characterized in that the method for detecting fluorescence from a liquid sample housed in a transparent or translucent sample container comprises providing a transparent or translucent sample container in a sample holder, which is opaque except for a sample container introduction opening, an excitation light incoming opening and a fluorescence outgoing opening; layering a liquid sample and a shielding liquid unmixable therewith to prevent external light from entering through the sample container introduction opening; introducing excitation light from such a direction that the excitation light can irradiate the liquid sample before irradiating the shielding liquid; and detecting fluorescence, which emits in a direction of avoiding absorption by the shielding liquid.
The fluorescence detection method according to a second aspect of the present invention, which is provided to attain the object, is characterized in that the shielding liquid is made of oil with a shielding agent added thereto in the method according to the first aspect.
The fluorescence detection method according to a third aspect of the present invention, which is provided to attain the object, is characterized in that the shielding liquid is made of carbon black in the method according to the second aspect.
The fluorescence detection system according to a fourth aspect of the present invention, which is provided to attain the object, is characterized in that the fluorescence detection system comprises a sample holder for firmly holding a plurality of sample containers so as to be spread and held along an arc; a partition plate coupled to a drive unit so as to be rotatable about a center of the arc; an excitation light source, an optical unit for excitation light and an optical unit for fluorescence, which are fixedly provided to the partition plate so as to be integrally rotatable; and an optical sensor, which is fixedly provided in mechanically independent fashion with respect to the rotatable drive unit; wherein
(a) excitation light intensity from the excitation light source is modulated so as to have a constant frequency;
(b) the optical unit for excitation light is provided so as to selectively excite a single sample container by directing excitation light from the excitation light source to the selected sample container;
(c) the optical unit for fluorescence includes an optical guide for transmitting a fluorescent signal from the selected sample container to the optical sensor, an incoming end of the optical guide can be provided so as to confront the selected sample container with the excitation light being directed thereto to pick up the fluorescent signal, and an outgoing end of the optical guide can be provided so as to confront the optical sensor on a center of rotation of the drive unit;
(d) the excitation light is directed to the respective sample containers spread along the arc one after another by rotation of the partition plate, and simultaneously fluorescence is directed to the optical sensor through the optical unit for fluorescence including the optical guide; and
(e) an electrical signal outputted from the optical sensor is phase-detected by a modulated frequency of excitation light intensity.
The fluorescence detection system according to a fifth aspect of the present invention, which is provided to attain the object, is characterized in that the optical unit for excitation light is fixedly provided to the partition plate so as to selectively excite a single sample container by directing the excitation light from the excitation light source from beneath in an upward direction, and the optical unit for fluorescence is fixedly provided to the partition plate so as to pick up a fluorescent signal advancing from the sample container toward the center of rotation of the partition plate in a substantially horizontal direction in the fourth aspect.
The fluorescence detection system according to a sixth aspect of the present invention, which is provided to attain the object, is characterized in that the system according to the fourth aspect or the fifth aspect further comprises a thermostatic unit for controllably setting a sample at a desired temperature.